Crystalline vap-1 and uses thereof

ABSTRACT

The present invention relates to crystalline vascular adhesion protein-1 (VAP-1) and in particular to methods for the use of structural information of crystalline human VAP-1 for ligand and/or inhibitor identification, design and production, as well as screening assays for detections of same. The invention further relates to inhibitors identified by the assays according to the present invention.

This Application is a Divisional of co-pending application Ser. No.10/557,188, filed on Nov. 17, 2005, which was filed as a national stageapplication of International Application No. PCT/FI2004/000318 filed May25, 2004. This Application also claims priority under 35 U.S.C. §119 toApplication No. FI 20030786 filed on May 26, 2003 and Application No. FI20040271 filed on Feb. 20, 2004, both filed in Finland. The entirecontents of the aforementioned applications are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present Invention relates to crystalline human vascular adhesionprotein-1 (VAP-1) and in particular to the use of structural informationof crystalline human VAP-1 for ligand and/or inhibitor identification,design and production, as well as in silico and in vitro screeningassays for detection of such ligands and/or inhibitors. The inventionfurther relates to inhibitors identified by the assays according to thepresent invention, which are useful in the treatment of acute andchronic inflammatory conditions, diseases related to carbohydratemetabolism, aberrations in adipocyte or smooth muscle cell function andvarious vascular diseases.

BACKGROUND OF THE INVENTION

Physiological immune surveillance is dependent on the continuouspatrolling of lymphocytes between the blood and different lymphoidorgans. In normal non-lymphoid tissue lymphocytes are absent or onlypresent at a very low level, but in many inflammatory disease statesvast numbers of lymphocytes can accumulate in various affected tissuesand organs. One of the important molecules controlling lymphocyte exitfrom the blood is vascular adhesion protein-1 (VAP-1) disclosed in U.S.Pat. No. 5,580,780. VAP-1 is a homodimeric 170-180 kDa endothelialglycoprotein. VAP-1 mediates lymphocyte binding to venules in humantissue sections. VAP-1 is heavily glycosylated and the sugar moietiesare important for the adhesion function (Salmi et al., 1996). Blockingthe adhesive function of VAP-1 reduces the number of cells infiltratinginflamed tissue allowing the inflammation to resolve. VAP-1 is thus atarget for anti-inflammatory drug development.

Human vascular adhesion protein-1 (VAP-1) is a membrane-boundmultifunctional glycoprotein with both adhesive and enzymaticproperties. The cloning of VAP-1 surprisingly revealed that it belongsto the semicarbazide sensitive monoamine oxidases (SSAO; EC 1.4.3.6)(International Patent Publication WO 98/53049). VAP-1 is a type 2integral membrane protein with a large catalytically activeextracellular domain. Thus VAP-1 is an ectoenzyme. Neither the role ofSSAO activity nor its physiological substrates in leukocyte endothelialinteraction is well defined. VAP-1 was the first molecularly definedtransmembrane member of this enzyme group in mammals, and it accountsfor 90% of cellular SSAO activity. Notably, SSAOs are different from thewell characterized monoamine oxidases A and B in respect to subcellularlocalization, substrates, cofactors, inhibitors, and protein sequence.

Although the SSAO reaction has been known since the 1950's inbiochemical terms, the physiological function(s) of these enzymes hasremained enigmatic. The physiological substrates of SSAO are not known.Two potential candidates, methylamine and aminoacetone, are howeverformed during intermediary metabolism in humans and can be deaminated bySSAO in vitro and in vivo.

VAP-1 SSAO activity has been proposed to be directly involved in thepathway of leukocyte adhesion to endothelial cells by a novel mechanisminvolving direct interaction with an amine substrate presented on aVAP-1 ligand expressed on the surface of a leukocyte (Salmi et al.,2001). This publication describes the direct involvement of VAP-1 SSAOactivity in the process of adhesion of leukocytes to endothelium. Thusinhibitors of VAP-1 SSAO activity could be expected to reduce leukocyteadhesion in areas of inflammation and thereby reduce leukocytetrafficking into the inflamed region and therefore the inflammatoryprocess itself.

In human clinical tissue samples expression of VAP-1 is induced at sitesof inflammation. This increased level of VAP-1 can lead to increasedproduction of H₂O₂ generated from the action of the VAP-1 SSAOextracellular domain on monoamines present in the blood. This generationof H₂O₂ in the localised environment of the endothelial cell couldinitiate other cellular events. H₂O₂ is a known signalling molecule thatcan upregulate other adhesion molecules and this increased adhesionmolecule expression may lead to enhanced leukocyte trafficking intoareas in which VAP-1 is expressed. Other products of the VAP-1 SSAOreaction may also have biological effects also contributing to theinflammatory process. Thus the products of the VAP-1 SSAO activity maybe involved in an escalation of the inflammatory process, which could beblocked by specific SSAO inhibitors.

VAP-1 SSAO may be involved in a number of other pathological conditionsassociated with an increased level of circulating amine substrates ofVAP-1 SSAO. The oxidative deamination of these substrates would lead toan increase in the level of toxic aldehydes and oxygen radicals in thelocal environment of the endothelial cell which could damage the cellsleading to vascular damage. Increased levels of methylamine andaminoacetone have been reported in patients with Type I and Type IIdiabetes and it has been proposed that the vasculopathies such asretinopathy, neuropathy and nephropathy seen in late stage diabetescould be treated with specific inhibitors of SSAO activity.

The development of specific VAP-1 SSAO inhibitors that modulate VAP-1activity would be useful for the treatment of acute and chronicinflammatory conditions or diseases such as chronic arthritis,inflammatory bowel diseases, and skin dermatoses, as well as diseasesrelated to carbohydrate metabolism (including diabetes and complicationsresulting from diabetes, such as vasculopathies). In addition,aberrations in adipocyte differentiation or function and smooth musclecell function (in particular, atherosclerosis), and various vasculardiseases maybe suitable for treatment with VAP-1 SSAO inhibitors.

International Patent Publication WO 03/006003 discloses carbocyclichydrazino compounds as well as the use thereof as inhibitors ofsemicarbazide-sensitive amine oxidases (SSAO), including human VascularAdhesion Protein-1 (VAP-1).

Copper-containing amine oxidases (CAOs; EC 1.4.3.6) belong to thefunctionally diverse superfamily of amine oxidases (Dawkes et al.,2001). They are also known as semicarbazide-sensitive amine oxidasessince their enzymatic activity can be blocked by a carbonyl-reactivecompound, semicarbazide. They catalyse the oxidative deamination ofprimary amines to the corresponding aldehydes in a copper-dependentreaction where molecular oxygen is consumed and hydrogen peroxide andammonia are released. A characteristic feature for all CAOs is the useof 2,4,5-trihydroxyphenylalanine quinone, a topaquinone (T P Q), as aredox cofactor. CAOs have been isolated from several differentorganisms, including bacteria, fungi, plants and mammals. In plants CAOsare involved, e.g. in wound healing, whereas in prokaryotes CAOs allowthe organism to utilize various amines metabolically as sources ofnitrogen and carbon. In higher eukaryotes very little is known about thebiological function of CAOs besides their role in the metabolism ofbiogenic and other amines.

Shepard et al., 2002, report striking differences in selectivity andrates of inactivation when testing inhibitors against six known coppercontaining amine oxidases.

The crystal structures of CAOs have been solved from four differentspecies: Escherichia coli (ECAO; e.g. Protein Data Bank, PDB code 1 oac)(Parsons et al., 1995), Pisum sativum (PSAO; PDB code 1ksi) (Kumar etal., 1996), Hansenula polymorpha (HPAO; e.g. PDB code 1a2v) (Li et al.,1998) and Arthobacter globiformis (AGAO; e.g. PDB code 1av4) (Wilce etal., 1997). All of these homodimeric structures have a similar overallfold that can be divided into domains D1-D4 of which the D1 domain isfound only in E. coli. Domains D2 and D3 are ˜100 amino acids each andhave an α/β type fold, whereas the largest, C-terminal domain D4 is ˜400amino acids in length and has a unique β-sandwich fold that is neededfor dimerization. The active site, which is located in the D4 domain, ishighly conserved within the CAO family. It is buried deeply within theprotein and accessible only via a long channel surrounded mainly byamino acids from the D3 and D4 domains. The amino acid residues of theD3 domain are less conserved than the actual active site, suggestingthat the cavity leading to the active site is of great importance indetermining the substrate specificity of CAOs.

Even though the structures of known CAO proteins are quite similar, thesequence identity at the amino acid level is only 25-35%. Theevolutionary relationship of VAP-1 to the structurally known members ofthe CAO family has not been characterized, but the presence of atransmembrane domain at the N-terminus of VAP-1 suggests substantialdivergence from the soluble CAOs.

The present invention provides the crystallization and X-ray analysis ofhuman VAP-1. This is the first mammalian CAO to be crystallized.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to crystalline vascular adhesion protein-1(VAP-1), especially human VAP-1.

The present invention provides means and methods for crystallizingpurified VAP-1, analysing the obtained VAP-1 crystals, obtaining crystalparameters and X-ray diffraction data.

The present invention further relates to compositions comprisingcrystalline VAP-1 and the use of such compositions.

The present invention further provides structural information ofcrystalline human VAP-1, more specifically information on the activesite cavity of human VAP-1, wherein said cavity is ˜20 Å×˜10 Å wide atthe surface and ˜15 Å deep and amino acids 86-87, 97, 168-173, 176-177,180, 184, 205-212, 216, 227, 232-234, 236-239, 344, 388-390, 393-397,415-419, 421, 421-426, 467-470, 647-651 and 758-761 from one monomer,and amino acids 443-449 and 451 of the other monomer of human VAP-1.More specifically said active site further comprises amino acid Leu469at the top of a narrow ˜4.5×˜4.5 cavity at the bottom of the site, whichis lined by amino acid residues Ala370, Tyr384, Asp386, Asn470, Tpq471and Tyr473.

The present invention further provides a computer readable medium havingstored thereon the atomic co-ordinate/X-ray diffraction data definingthe three-dimensional structure of human VAP-1 protein, said mediumbeing capable of displaying a three dimensional representation of acrystal of a molecule comprising a fragment of human VAP-1 protein whenread by an appropriate machine and processed by a computer program fordetermining molecular structures.

The present invention further provides an in silico assay for de novodesign of ligands and/or inhibitors comprising (i) identification offunctional groups or small molecule fragments which can interact withsites in the active site of VAP-1, and (ii) linking these in a singlecompound.

The present invention further provides an in silico assay for screeningknown compounds and compound libraries for their ability of inhibitingVAP-1 activity.

The present invention further provides novel VAP-1 inhibitors useful forthe treatment of acute and chronic inflammatory conditions or diseasessuch as chronic arthritis, inflammatory bowel diseases, and skindermatoses, multiple sclerosis, as well as diseases related tocarbohydrate metabolism (including diabetes and complications resultingfrom diabetes, such as vasculopathies). In addition, such inhibitors maybe useful for treating aberrations in adipocyte differentiation orfunction and smooth muscle cell function (in particular,atherosclerosis), and various vascular diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention will be described in greater detail bymeans of preferred embodiments and with reference to the attachedfigures, in which

FIG. 1, Panel A shows a hexagonal VAP-1 crystal and Panel B a typicaldiffraction pattern;

FIG. 2 shows the dimer of the human VAP-1 crystal structure, wherein thesugar units are drawn as space-filling models and the Tpg471 in bothmonomers is shown as a sphere in the D4-domain;

FIG. 3 shows one of the monomers of the dimeric VAP-1 crystal structure;and

FIG. 4 shows the binding mode of a VAP-1 inhibitor compound in the VAP-1structure. The point of view is down from the surface towards the activesite.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides crystals of a mammalian copper containingamine oxidase, CAO, especially crystals of human VAP-1.

To grow the crystals of the present invention a full-length protein,including the N-terminal transmembrane region, has to be purified togreater than 80% total protein and more preferably to greater than 90%total protein, most preferably to greater than 95% total protein. Forexpression and purification purposes the full length VAP-1 encodingsequence (SEQ ID NO. 1) is used. It is important that the purificationmethod of choice is such that the purified protein retains its CAOactivity, which may be determined using benzylamine as substrate.

A large number of vector-host systems known in the art may be used forrecombinant production of the protein for the crystallization process.Possible vectors include, but are not limited to, plasmids or modifiedviruses, but the vector system must be compatible with the host cellused. As human VAP-1 is heavily glycosylated, eukaryotic hosts, such asyeast or animal cell hosts are preferred. Chinese Hamster Ovary (CHO)cells constitute most preferred host cells, these cells being fullycapable of glycosylation.

Any crystallization technique known to those skilled in the art may beemployed to obtain the crystals of the present invention, including, butnot limited to, batch crystallization, vapour diffusion and microdialysis. Standard micro and/or macro seeding of crystals may be used ifrequired to obtain X-ray quality crystals.

The crystals of the present invention may form in the space group P6₅22with two molecules in the asymmetric unit and with unit dimensions ofa=b=225.9 Å, c=218.7 Å, α=β=90°, γ=120° (see Example 2 below). However,the present invention contemplates crystals, which form in any spacegroup including, but not limited to, P6₅22. The crystals diffract to aresolution greater than 4 Å, preferably greater than 3 Å, mostpreferably greater than 2.8 Å.

To collect diffraction data from the crystals of the present invention,the crystals may be protected using cryoprotectants, such as glycerol,and flash-frozen in a nitrogen stream. The X-ray diffraction data may beprocessed with the XDS-program (Kabsch et al., 1993), but any methodknown to those skilled in the art maybe used to process the X-raydiffraction data.

In order to determine the atomic structure of human VAP-1 according tothe present invention, molecular replacement (MR), model building andrefinement may be performed.

For determination of the VAP-1 structure molecular replacement may beemployed using the known structures of the CAOs known in the art or anyother CAO structure which may be determined as described above and belowin Example 3.

Any method known to the skilled artisan may be employed to determine thethree-dimensional structure of the protein of the present invention bymolecular replacement. For example, the program AMORE of the CCP4iprogram suite may be employed to determine the structure of human VAP-1using the atomic co-ordinates derived as described herein.

The atomic coordinates may be provided on a computer readable medium.Such a storage medium is preferable a random-access memory (RAM), aread-only memory (e.g. a CD-ROM) or diskette. The storage medium may beon a locally accessible computer, or remotely accessible through theinternet.

An initial model of the three dimensional structure may be built usingthe program O (Jones et al., 1991) and refined using e.g., the REFMACprogram of the CCP4i program suite.

The refined three-dimensional VAP-1 structure according to the presentinvention is represented by the atomic coordinates and the structuredetermination statistics given in Table 1. The refined X-ray structureof VAP-1 dimer consists of residues A55-A761 with residues A1-A54,A202-A204 and A762-A763 not modelled in monomer A and of residuesB57-B761 with residues B1-B56, B203, B742-B746 and B762-B763 notmodelled in monomer B. Residues, A471 and B471 are topaquinones(Tpq471), which are formed by post-translational modification of anintrinsic tyrosine residue and are involved in the catalytic reaction.Both of the monomers contain one copper ion in the active site.

The information obtained from the three-dimensional structure of thepresent invention reveals that human VAP-1 active site (the substratebinding site) comprises (1) three histidines, His520, His522 and His684,coordinating the copper ion, (2) a catalytic base, Asp386, as well as(3) Tyr372 and Asn470, which is involved in positioning Tpq471, and (4)the active site gate residue, Tyr384. Tpq471 is in the ‘on-copper’(inactive) conformation in the VAP-1 X-ray structure. Three-dimensionalrepresentations of the human VAP-1 structure are given in FIGS. 2 and 3.

The VAP-1 active site is deeply buried and Tpq471 is located ˜22 Å fromthe surface of the molecule. The active site cavity is ˜20 Å×˜10 Å wideat the surface and ˜15 Å deep. At the bottom of the cavity Leu469, whichis located on top of Tpq471, blocks the entrance to the active site inthe VAP-1 X-ray structure. One wall of the cavity is composed ofresidues from the smaller D2 and D3 domains comprising e.g. 86-87, 97,168-173, 176-177, 180, 184, 205-212, 216, 227, 232-234 and 236-239 andresidues from the long β-hairpin arm protruding from the other monomercomprising 443-449 and 451. The other wall of the channel is composed ofresidues from the catalytic D4 domain comprising e.g. 344, 388-390,393-397, 415-419, 421, 421-426, 467-470, 647-651, 758-761. At theentrance of the active site channel one sugar unit of the N-glycanattached to Asn232 is visible in the X-ray structure. Below Leu469 the˜7 Å long active site cavity is much narrower than at the surface andalmost circular with dimensions of ˜4.5×4.5 Å. This part of the channelis lined by residues Ala370, Tyr384, Asp386, Asn470, Tpq471 and Tyr473.

VAP-1 X-ray structure surprisingly reveals an unique structure of theactive site cavity. The cavity is extremely wide-mounted and opencompared to the narrow active site channels in the ECAO, HPAO, PSAO andAGAO structures. Therefore, the active site cavity of VAP-1 canaccommodate much larger ligand and inhibitor structures than the activesite cavities of ECAO, HPAO, PSAO and AGAO. The residue corresponding toLeu469 in VAP-1 is glycine (ECAO, HPAO and AGAO) or alanine (PSAO) and,thus, it cannot block the active site in the ECAO, HPAO, PSAO and AGAOstructures. Knowledge of this unique structure is necessary fordesigning a pharmacophore of human VAP-1 and specific ligand andinhibitor structures fitting the cavity.

This information can be used to define a pharmacophore of human VAP-1,i.e. a collection of chemical features and three-dimensional constraintsexpressing specific features necessary for biological activity. Thepharmacophore preferably includes surface-accessible features, such ashydrogen bond donors and acceptors, charged groups or hydrophobic sites.Such features may be included in a pharmacophore model based on theirrelative importance to the biological activity.

Pharmacophores may be determined using available computer software, suchas CATALYST, CERIUS or by using manual modelling based on knownconformation of lead compounds. The pharmacophore may be used to screenin silico compound libraries, using available computer software, asdescribed in more detail below.

In one embodiment of the invention the molecular modelling techniquesare thus used for de novo compound design. De novo compound designrefers to a process, where binding surfaces or active sites of targetmacromolecules, such as VAP-1, are determined and used as a basis for arational design of compounds that interact with said binding surface oractive site. The molecular modelling steps according to the presentinvention make use of the atomic coordinates of human VAP-1 and modelsof the active site. In a preferred embodiment the de novo drug designinvolves the identification of functional groups or small moleculefragments which can interact with the active sites in the bindingsurface of human VAP-1, and linking these groups or fragments into asingle compound. Once functional groups, or small molecule fragmentswhich interact with the specific active site of human VAP-1 have beenidentified, these can be linked into a single compound using eitherbridging fragments having suitable size and geometry to fit the activesite.

While linking of suitable functional groups and fragments may beperformed manually, it is preferred to use suitable software, such asQUANTA or SYBYL. Further software known in the art are e.g., HOOK, whichlinks multiple functional groups with molecular templates from adatabase, and CAVEAT, for designing linking units to constrained acyclicmolecules. Other computer-based approaches for de novo compound designinclude LUDI, SPROUT and LEAP-FROG.

The present invention permits the use of molecular design techniques todesign, identify and synthesize chemical entities and compounds,including inhibitory compounds, capable of binding to the active site ofhuman VAP-1. The atomic coordinates of human VAP-1 may be used inconjunction with computer modelling using a docking program, such ase.g., GOLD, GRAM, DOCK, HOOK or AUTODOCK, to identify potentialinhibitors of human VAP-1. This procedure can include computer fittingof potential inhibitors to the active site of VAP-1 to ascertain howwell the shape and the chemical structure of the potential inhibitorwill complement the active site. Examples of potential inhibitorsdesigned by modelling with a docking program may conform to the generalformula (I) as described below.

The present invention further includes an in silico method foridentifying compounds that interact with the active site of human VAP-1,comprising the steps of (a) providing the atomic co-ordinates of theligand binding domain of human VAP-1 in a storage medium on a computer,and (b) using the computer to apply molecular modelling techniques tothe co-ordinates.

The above described structure model was further used for testing alibrary of molecules built and energy-minimized with Sybyl v.6.9 toassess their ability of acting as VAP-1 inhibitors. Prior to the insilico screening the VAP-1 X-ray structure was modified to mimic the‘off-copper’ active conformation of CAOs as described in Example 4.

In a further embodiment of the present invention the in silico methodis, used for identifying compounds that specifically inhibit theenzymatic activity of human VAP-1, characterized by the following steps;providing a compound identified by said molecular modelling techniques,contacting said compound with human VAP-1 and detecting the interaction.

The three-dimensional structural information and the atomic coordinatesassociated with said structural information of VAP-1 is useful inrational drug design providing for a method of identifying inhibitorycompounds which bind to and inhibit the enzymatic activity of VAP-1.Said method for identifying said potential inhibitor for an enzymehaving SSAO-activity, comprises the steps of (a) using athree-dimensional structure of VAP-1 based on its atomic coordinateslisted in Table 1; (b) employing said three-dimensional structure todesign or select said potential inhibitor; (c) synthesizing saidpotential inhibitor; (d) contacting said potential inhibitor with saidenzyme; and (e) determining the ability of said inhibitor to inhibitsaid SSAO activity.

The invention further encompasses compounds identified using the presentin silico method for identifying compounds inhibiting the enzymaticactivity of human VAP-1, or compounds that interacts with the activesite of VAP1.

Examples of potential VAP-1 inhibitors identifiable by the method of thepresent invention may be represented by formula (I)

whereinR¹ is hydrogen, lower alkyl or an optionally substituted phenyl orheteroaryl group;R² is hydrogen or lower alkyl, orR¹ and R² may form together with the nitrogen atom to which they areattached a saturated heterocyclic ring;R³-R⁵ represent each independently hydrogen, lower alkyl, aralkyl,optionally substituted phenyl or a heteroaryl group, orR² and R³ may form together with the atoms to which they are attached asaturated heterocyclic ring, orR³ and R⁵ may form together with the carbon atoms to which they areattached a saturated carbocyclic ring;R⁶ is naphtyl, phenyl, substituted phenyl or a heteroaryl group;R⁷ is hydrogen, lower alkyl or aralkyl;n is 1, 2 or 3; and

X═0, S, SO, SO₂ or NR².

Other types of compounds may equally well be identified using themethods and models according to the present invention. A man skilled inthe art may, based on the atomic coordinates and the pharmacophoredescribed herein, identify compounds that interact with the active siteof the human VAP-1 protein. Generally, such potential inhibitorcompounds may be characterized by a carbonyl group reactive agent, suchas an amine or a hydrazine, as exemplified above, but other structureswith a narrow protruding part fitting into the 4.5×4.5 Å bottom part ofthe active site cavity and capable of binding to the TPQ at the bottomof the active site are equally potential inhibitors. This protrudingpart of the compounds according to the present invention, is connectedto a relatively hydrophobic middle part of about 7 Å length of thecompound, acting both as a “linker”, allowing the access of theprotruding part to the active site, and interacting with amino acids Ala370, Tyr384, Asp386, Asn470, Tpq471 and Tyr473 lining the cavity.Finally, the compounds according to the present invention comprise abuilder part that fills the wide cavity and interacts with theexemplified amino acids 86-87, 97, 168-173, 176-177, 180, 184, 205-212,216, 227, 232-234, 236-239, 344, 388-390, 393-397, 415-419, 421,421-426, 467-470, 647-651, 758-761, 443-449 and 451 in the walls of theactive site cavity.

Furthermore, the present invention provides assays and means forverifying the expected activity of the identified compounds.

The invention further encompasses the use of compounds identified by thepresent screening assays for the preparation of medicaments for thetreatment of acute and chronic inflammatory conditions or diseases suchas chronic arthritis, inflammatory bowel diseases, skin dermatoses andmultiple sclerosis, as well as diseases related to carbohydratemetabolism (including diabetes and complications resulting fromdiabetes, e.g., vasculopathies, such as retinopathy, nephropathy andneuropathy). In addition, such inhibitors may be useful for treatingaberrations in adipocyte differentiation or function and smooth musclecell function (in particular, atherosclerosis), and various vasculardiseases.

The invention further encompasses pharmaceutical compositions containingcompounds identified by the present screening assays.

The following examples illustrate the present invention.

Example 1 Production and Purification of Human VAP-1

The full-length protein with the N-terminal transmembrane region wasexpressed in glycosylation-competent CHO cells, as described in Smith etal., 1998. The harvested cells were lysed using a lysis buffer (150 mMNaCl, 10 mM Tris-Base pH 7.2, 1.5 mM MgCl2, 1% NP40). Clarified celllysate was used for the purification of HVAP-1 based on a monoclonalantibody affinity column and using the ÄKTA™ purifier system (AmershamBiotech). The protein was purified to homogeneity (>95%) using affinitychromatography and after purification the presence of the VAP-1 protein,the 90- and 170-180-kD bands, was confirmed by silver stained SDS-pageas described by Smith et al. 1998.

The purified protein retained its CAO activity as determined usingbenzylamine as the substrate Amine oxidase activity was measured using aspectrophotometric method as described (Holt et al., 1997), 200 μlvolume and 1 mM benzylamine as the substrate. The absorbance change wasmonitored in a Victor' multi-label plate counter at 490 nm (PerkinElmerLife Sciences).

The nucleotide sequence of the coding region of human VAP-1 is given inthe sequence listing as SEQ ID NO. 1, and corresponding amino acidsequence (763 aa:s) as SEQ ID NO.2.

Example 2 Crystallization and Preliminary Analysis

Initial crystallization conditions for hVAP-1 were screened at roomtemperature using the Wizard I random sparse matrix crystallizationscreen (Emerald BioStructures, Inc., USA) and the vapour-diffusionmethod. Small hexagonal crystals were obtained in a condition containing1.0 M K/Na tartrate, 100 mM imidazole (pH 8.0) and 200 mM NaCl afterseveral months of incubation. The hanging drops contained 2 μl ofprotein sample (1.0 mg/ml) in 10 mM potassium phosphate buffer (pH 7.2)and 2 μl of reservoir solution. After optimization the best crystalswere obtained using a reservoir solution of 1.0 M K/Na tartrate, 100 mMimidazole (pH 7.8) and 150-250 mM NaCl as the precipitant. The crystalsformed in a few days and grew to a final size of about 0.15×0.15×0.1 mm(FIG. 1).

One crystal was mounted in a capillary and preliminary X-ray analysiswas carried out in-house using a rotating-anode radiation source (Cu Kαradiation, 50 kV, 150 mA) and MAR345 image plate detector. The crystal,however, diffracted to only 8 Å resolution and the space group could notbe accurately determined. All further X-ray analysis, as well as datacollection, was carried out using synchrotron radiation at the beamlineX11 (EMBL/DESY Hamburg, Germany) equipped with a MAR Research CCDdetector. For data collection, the crystals were cryo-protected with 20%(v/v) glycerol and flashfrozen in a 100 K nitrogen stream. Diffractiondata, collected from three different crystals, were processed with theprogram XDS. The solvent content and Matthews coefficient werecalculated assuming a molecular weight of 90 kDa per monomer and usingthe CCP4 suite.

The best looking, hexagonal crystals were obtained using 1.0 M K/Natartrate, 100 mM imidazole (pH 7.8) and 150-250 mM NaCl as theprecipitant. The crystals grew to a typical size of 0.15×0.15×0.1 mm(FIG. 1 a) with a unit cell dimension of a, b=225.9 Å, c=218.7 Å, α,β=90° and γ=120°. According to the diffraction data statistics (Table 1)the diffraction limit of the VAP-1 crystals was 3.2 Å, even thoughreflections corresponding to greater than 3.0 Å resolution were observedin some frames (FIG. 1 b). The crystals belong to space group P6₅22.Assuming the presence of one dimer (180 kDa) per asymmetric unit, theMatthews coefficient is 4.5 Å³/Da and solvent content 72%. Crystalparameters and diffraction data statistics are summarized in Table 1.

TABLE 1 Crystal and diffraction data statistics. Space group p6₅22^(a)Unit cell lenghts (Å) a = b = 225.9, c = 218.7 Unit cell angles (°) α, β= 90, γ = 120 Matthews coefficient (Å³ Da⁻¹) 4.5^(a) Solvent percentage72.3^(a)  Unit cell volume (Å³) 9665176 Molecules per asymmetric unit2^(a)   Unique reflections 52367 (4588)  Observed reflections 739050(61793)  Wavelength used (Å)  0.811 Resolution range (Å)   20-3.20(3.30-3.20) Completeness (%) 95.9 (97.2) R_(merge)(%) 19.6 (46.4)Average I/σ 13.2 (6.0)  Redundancy 14.1 (13.5) Values in parenthesisrefer to the highest resolution shells. ^(a) See text for details.

Example 3 Structure Determination

The structure of VAP-1 was solved by molecular replacement using theprogram AMORE (Navaza, 1994) of the CCP4i program suite (CollaborativeComputational Project, 1994). This method confirmed that the space groupof VAP-1 crystals was P6₅22 with one biological unit, a dimer perasymmetric unit. Out of the dimeric polyalanine backbones of Escherichiacoli CAO (residues 93-720; PDB code 1OAC), Pisum sativum CAO (residues7-634; PDB code 1KSI), Hansenula polymorphs CAO (residues 22-655; PDBcode 1A2V) and Arthobacter globiformis CAO (residues 9-623; PDB code1AV4) tested in molecular replacement, the structure of P. sativum gavethe best correlation coefficient (44.1%) and Rfactor (53.4%) and wasused a search model.

Electron density maps were calculated with FFT of CCP4i suite and werepredictable enough to trace the VAP-1 polypeptide, even though, at thebeginning of building, in several fragments. The model was manuallybuilt using the program O and refined with REFMAC 5.1.24 (Murshudov etal., 1997) of the CCP4i suite to 3.2 Å resolution.

Side chains were step by step added to the structure between thenumerous cycles of refinement. In the final model, only the followingamino acids could not be traced and, thus, are not included in thestructure: A1-A54, A202-A204, A762-A763, B1-B56, B203, B742-B746 andB762-B763. Coordinates for the topa-quinone residue were taken fromHetero-compound Information Centre, Uppsala (Kleywegt and Jones, 1998)and dictionary for it was generated with the program PRODRG (van Aaltenet al., 1996). The stereochemical quality of the final VAP-1 model wasassessed with PROCHECK (Laskowski et al, 1993)—out of the 1401 aminoacids in the model, 84.0% occurred in the most favored regions in theRamachandran plot (Ramachandran and Sasisekharan, 1968) and only 0.5% inthe generously allowed or disallowed regions. A summary of the structuredetermination statistics is presented in Table 1. The atomic coordinates(and the structure factors) for the human VAP-1 crystal structure havebeen deposited in the PDB with the entry code 1PU4 and 1US1.

The crystalline form of human VAP-1 is a homodimer, each subunitcontaining the domains D2, D3 and D4. A schematic representation of theVAP-1 structure is shown in FIG. 2. The D2, D3 and D4 domains seen inthe crystal structure, consist of residues ˜55-169, 170-300 and 301-761,respectively (SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5,respectively). The major differences in the structure of human VAP-1compared to the other known structures of amine oxidases are seen in thedomains D2 and D3, even though there are differences also in the domainD4.

Example 4 In Silico Method for Screening Potential-Inhibitors

This example shows how a library of aryloxymethyl-substituted hydrazinealcohol compounds is screened in silico by docking into the bindingcavity of VAP-1 described in Example 3. The compounds screened have theformula

whereinR¹ is hydrogen, lower alkyl or an optionally substituted phenyl orheteroaryl group;R² is hydrogen or lower alkyl, orR¹ and R² may form together with the nitrogen atom to which they areattached a saturated heterocyclic ring;R³-R⁵ represent each independently hydrogen, lower alkyl, aralkyl,optionally substituted phenyl or a heteroaryl group, orR² and R³ may form together with the atoms to which they are attached asaturated heterocyclic ring, orR³ and R⁵ may form together with the carbon atoms to which they areattached a saturated carbocyclic ring;R⁶ is naphtyl, phenyl, substituted phenyl or a heteroaryl group;R⁷ is hydrogen, lower alkyl or aralkyl;n is 1, 2 or 3; and

X═O, S, SO, SO, or NR².

Examples of compounds synthesized and/or built and screened in silicoare given in Table 2:

TABLE 2 Compound Structure 1

3

4

12

7

16

2

6

8

11

5

14

10

9

13

19

17

20

Modification of the X-Ray Structure and Ligand Docking

The VAP-1 X-ray structure was modified to mimic the ‘off-copper’ activeconformation of CAOs as described below. Firstly, topaquinone wasmodified to the active imino-quinone form according to the ECAOstructure (PDB code 1D6Z (Wilmot et al., 1999)). Secondly, the activesite cavity of the VAP-1 structure was modified prior to docking studiesby choosing side chain rotamers (Phe389, Tyr394, Asp386 and Leu469) inthe Bodil modeling package(www.abo.fi/fak/innf/bkf/research/johnson/bodil.html) that made theactive site imino-quinone more accessible for the ligands. Hydrogenatoms were added to the VAP-1 structure used for screening and dockingwith the program Reduce v.2.15 without any side chain adjustment (Wordet al., 1999).

R- and S-enantiomers of potential ligands were built and energyminimized with Sybyl v.6.9 (Tripos Associates, St. Louis, USA). Theligands were covalently bound to the imino-quinone residue and manuallydocked into the binding cavity.

Ligand Binding Mode

FIG. 4 shows an example of the ligand screened. In FIG. 3, which is madeusing the Bodil modeling package, the binding mode of the S-enantiomerof BTT-2071 (Compound 9) is presented. The S-enantiomer of BTT-2071 waschosen to be an example ligand in the figure, since based on the dockingsimulations it is able to form the most extensive interactions with theVAP-1 structure.

Based on the VAP-1 structure, ligands where X is a sulphur atom insteadof oxygen atom interact more favorably with VAP-1 since the interactionsurface in VAP-1 is hydrophobic.

The hydrophobic part of the ligands including the sulphur atom packsagainst hydrophobic residues (Leu468, Leu469, Phe389 and Met211). Theoxygen atom in the para-methoxyl group of the ligand is at the hydrogenbonding distance to the side chain oxygen of Thr212 whereas the oxygenatom in the meta-methoxyl group of the ligand is at the hydrogen bondingdistance to the side chain hydroxyl group of Tyr394. Phe389 is locatedat a position where it can easily interact with the aromatic ring in theligands. The methyl groups in the para- and meta-methoxyl groups are incontact with Tyr448 and Tyrl 76, respectively. The hydroxyl group in theS-enantiomers can form an optimal hydrogen bond with Asp386 whereas inthe R-enantiomers the hydrogen bonding distance and geometry are notoptimal.

Example 5 In Vitro Assay for Verifying VAP-1 Inhibitory Effect of theIdentified Potential Inhibitors

VAP-1 SSAO activity was measured using the coupled colourimetric methodessentially as described for monoamine oxidase and related enzymes.Recombinant human VAP-1 SSAO expressed in Chinese Hamster Ovary (CHO)cells was used as a source of VAP-1 SSAO for activity measurements.Native CHO cells have negligible SSAO activity. These cells and theirculture have previously been described (Smith D J et al., 1998).

A cell lysate was prepared by suspending approximately 3.6×108 cells in25 ml lysis buffer (150 mM NaCl, 10 mM Tris-Base pH 7.2, 1.5 mM MgCl2,1% NP40) and incubating at 4° C. overnight on a rotating table. Thelysate was clarified by centrifugation at 18000 g for 5 min at roomtemperature and the supernatant used directly in the assay.

The VAP-1 SSAO assay was performed in 96 well microtitre plates asfollows. To each well was added a predetermined amount of inhibitor ifrequired. The amount of inhibitor varied in each assay but was generallyat a final concentration of between 1 nM and 50 μM. Controls lackedinhibitor. The inhibitor was in a total volume of 20:1 in water. Thefollowing reagents were then added. 0.2M potassium phosphate buffer pH7.6 to a total reaction volume of 2000 μl, 45 μl of freshly madechromogenic solution containing 1 mM 2,4-dichlorophenol, 500 μM4-aminoantipyrine and 4 U/ml horseradish peroxidase and an amount of CHOcell lysate containing VAP-1 SSAO that caused a change of 0.6 A490 perh. This was within the linear response range of the assay.

The plates were incubated for 30 min at 37° C. and the backgroundabsorbance measured at 490 um using a Wallac Victor II multilabelcounter. To initiate the enzyme reaction 20 μl 10 mM benzylamine (finalconcentration=1 mM) was added and the plate incubated for 1 h at 37° C.

The increase in absorbance, reflecting VAP-1 SSAO activity, was measuredat 490 nm. Inhibition was presented as percent inhibition compared tocontrol after correcting for background absorbance and IC50. valuescalculated using GraphPad Prism.

Example 6 Comparison of YAP-1 SSAO Activity Versus Total Rat MAOActivity

Rat MAO was prepared from rat liver by rinsing a 1 g liver sampleseveral times in 14 ml KCl-EDTA-solution to remove all blood. Then 1 gliver sample was homogenised in 4 ml ice-cold potassium phosphate buffer(0.1 M, pH 7.4) with an Ultra-Turrax homogeniser (setting 11 000 rpm,4×10s). After centrifugation at 500 g for 10 min at 4° C. thesupernatant was carefully withdrawn and was centrifuged at 12 300 g for15 min at 4° C. The supernatant was discharged and sedimentedmitochondria were resuspended in 4 ml fresh phosphate buffer andcentrifuged as previously. The mitochondria were suspended in 4 mlphosphate buffer and homogenized with an Ultra-Turrax homogeniser(setting 11 000 rpm, 2×10s). Mitochondria) preparate was aliquoted andstored at −70° C.

Total MAO activity was measured in a similar way as for VAP-1 SSAOexcept that 2,4-dichlorophenol was replaced by 1 mM vanillic acid. Toeach well was added a predetermined amount of inhibitor if required. Theamount of inhibitor varied in each assay but was generally at a finalconcentration of between 10 nM and 800 mM. Controls lacked inhibitor.The inhibitor was in a total volume of 20:1 in water. The followingreagents were then added 0.2 M potassium phosphate buffer pH 7.6 for atotal reaction volume of 300 μl, 50 μl of freshly made chromogenicsolution (as above) and 50 μl of MAO preparation.

The plates were incubated for 30 min at 37° C. and the backgroundabsorbance measured at 490 nm using a Wallac Victor II multilabelcounter. To initiate the enzyme reaction 20 μl of 5 mM tyramine (finalconcentration 0.5 mM) was added and the plate incubated for 1 h at 37°C. The increase in absorbance, reflecting MAO activity, was measured at490 nm. Inhibition was presented as percent inhibition compared tocontrol after correcting for background absorbance and IC50 valuescalculated using GraphPad Prism. Clorgyline and pargyline (inhibitors ofMAO-A and -B respectively) at 0.5 μM were added to some wells aspositive controls for MAO inhibition.

The ability of compounds of Table 3 to inhibit VAP-1 SSAO activity withspecificity for VAP-1 SSAO over rat MAO is shown in Table 2. The resultsindicate that the compounds of the invention are specific inhibitors ofhuman VAP-1 SSAO activity. The compounds of the present invention aretherefore expected to have therapeutic utility in the treatment ofdiseases and conditions in which the SSAO activity of the human adhesionmolecule VAP-1 plays a role.

TABLE 3 Potency and specificity of the compounds tested Total MAO VAP-1SSAO in inhibitory Selectivity for inhibitory activity activity VAP-1SSAO over Compound IC₅₀ nM IC₅₀ nM MAO  1 (BTT-2066) 0.37 13 35  4 0.4310 23  7 0.44 6.0 14 10 0.55 6.0 11 13 0.64 3.3 5 16 0.35 7.4 21  2 0.528.7 17  5 0.52 4.0 8  8 0.65 10 15 11 (BTT-2067) 0.27 11 41 14 0.37 3.49 17 0.43 6.1 14  3 0.26 3.3 13  6 0.09 3.3 37  9 (BTT-2071) 0.21 41 19512 (BTT-2072) 0.33 37 112 15 (BTT-2072), 0.34 32 94 S-enantiomer 18(BTT-2072), 0.39 21 62 R-enantiomer 19 (BTT 2073) 0.20 8.8 45 20 0.349.6 28

It will be obvious to a person skilled in the art that, as thetechnology advances, the inventive concept can be implemented in variousways. The invention and its embodiments are not limited to the examplesdescribed above but may vary within the scope of the claims.

REFERENCES

-   Collaborative Computational Project, Number 4. (1994). Acta Cryst,    D50, 760763.-   Dawkes, H. C. & Phillips, S. E. (2001). Curt Opin Struct Biol, 11,    666-673.-   Holt, A., Sharman, D. F., Baker, G. B. & Paicic, M. M. (1997). Anal    Biochem, 244, 384-392.-   Kabsch, W. (1993). Journal of Applied Crystallography, 26, 795-800.-   Kumar, V., Dooley, D. M., Freeman, H. C., Guss, J. M., Harvey, L,    McGuirl, M A., Wilce, M. C. & Zubak, V. M. (1996). Structure, 4,    943-955.-   Li, R., Klinman, J. P. & Mathews, F. S. (1998). Structure, 6,    293-307.-   Parsons, M. R., Convery, M A, Wilmot, C. M., Yadav, K. D., Blakeley,    V., Corner, A. S., Phillips, S. E., McPherson, M. J. &    Knowles; P. F. (1995). Structure, 3, 1171-1184.-   Salmi, M. & Jalkanen, S. (1996). J Exp Med, 183, 569-579.-   Salmi, M. & Jalkanen, S. (2001). Trends Immunol, 22, 211-216.-   Smith, D. J., Salmi, M., Bono, P., Hellman, J., Leu, T. &    Jalkanen, S. (1998). J Exp Mad, 188, 17-27.-   Wilce, M. C., Dooley, D. M., Freeman, H. C., Guss, J. M., Matsunami,    H, McIntire, W. S., Ruggiero, C. E., Tanizawa, K. & Yamaguchi, H.    (1997). Biochemistry, 36, 16116-16133.-   Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard (1991). Acta    Crystallogr A47 (Pt 2), 110-119.-   Kleywegt, G. J., and Jones, T. A. (1998). Acta Cryst D 54,    1119-1131.-   Laskowski, R. A., Macarthur, M. W., Moss, D. S., and Thornton, J. M.    (1993). Journal of Applied Crystallography 26, 283-291.-   Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997). Acta    Crystallogr D 53, 240-255.-   Navaza, J. (1994). Amore—an Automated Package for Molecular    Replacement. Acta Cryst A 50, 157-163.-   Ramachandran, G. N., and Sasisekharan, V. (1968). Adv Protein Chem    23, 283-438.-   Shepard, E. M., Smith, J., Bradley, O. E., Kuchar, J. A.,    Lawrence, M. S, and Dooley, D. M. (2002). Eurl J. Biochem. 269,    3645-3658.-   van Aalten, D. M., Bywater, R., Findlay, J. B., Hendlich, M.,    Hooft, R. W., and Vriend, G. (1996). J Comput Aided Mol Des 10,    255-262.-   Wilmot, C. M., Hajdu, J., McPherson, M. J., Knowles, P. F., and    Phillips, S. E. (1999). Science 286(5445), 1724-8.-   Word, J. M., Lovell, S. C., Richardson, J. S., and    Richardson, D. C. (1999) J Mol Biol 285, 1735-47.

1. Crystalline human vascular adhesion protein 1 (VAP-1), wherein saidcrystal is defined as a crystal of space group P6522 with two moleculesin the asymmetric unit and with unit dimensions of a=b=225.9 Å, c=218.7Å, a=b=90°, g=120°.
 2. The crystalline VAP-1 according to claim 1, or ahomolog thereof, comprising domains D2, D3 and D4, characterized byamino acid sequences SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5,respectively.
 3. The crystalline VAP-1 protein according to claim 2,comprising an active site cavity which is ˜20 Å×˜10 Å wide at thesurface and ˜15 Å deep.
 4. The crystalline VAP-1 protein according toclaim 3, wherein said active site cavity comprises amino acids 86-87,97, 168-173, 176-177, 180, 184, 205-212, 216, 227, 232-234, 236-239,344, 388-390, 393-397, 415-419, 421, 421-426, 467-470, 647-651 and758-761 of SEQ ID NO. 2 from one monomer, and amino acids 443-449 and451 of SEQ ID NO. 2 from the other monomer of human VAP-1.
 5. Thecrystalline VAP-1 protein according to claim 4, wherein said active sitefurther comprises amino acid Leu469 at the top of a narrow ˜4.5 Å×˜4.5 Åcavity at the bottom of said cavity.
 6. The crystalline VAP-1 proteinaccording to claim 5, wherein said bottom part of the active site cavityis lined by amino acid residues Ala370, Tyr384, Asp386, Asn470, Tpq471and Tyr473.
 7. A composition comprising a crystalline VAP-1 according toclaim
 1. 8. A method for crystallizing human VAP-1, comprising the stepsof: a) providing an aqueous, solution comprising human VAP-1 protein; b)providing a reservoir solution comprising a precipitating agent; c)mixing a volume of said aqueous solutions with a volume of saidreservoir solution forming a mixed solution; and d) crystallizing atleast a portion of said mixed solution.
 9. The method of claim 8,wherein said aqueous solution provided in step a) has a concentration ofVAP-1 of 1 mg/ml.
 10. The method of claim 9, wherein the precipitatingagent is K/Na tartrate.
 11. The method of claim 10, wherein step d) isperformed by vapour diffusion.
 12. A computer readable medium comprisinga data storage material en-coded with machine readable data havingstored thereon atomic co-ordinate/X-ray diffraction data defining thethree-dimensional structure of human VAP-1 protein, capable ofdisplaying a three dimensional representation of a crystal of a moleculecomprising a fragment of human VAP-1 protein when read by an appropriatemachine and processed by a computer program for determining moleculestructures, wherein said data defines the active site cavity of dimerichuman VAP-1 protein and said active site cavity is ˜20 Å×˜10 Å wide atthe surface and ˜15 Å deep and further comprises amino acid Leu469 atthe top of a narrow ˜4.5×˜4.5 cavity at the bottom of the site.
 13. Thecomputer readable medium according to claim 12, wherein said active sitecavity comprises amino acids 86-87, 97, 168-173, 176-177, 180, 184,205-212, 216, 227, 232-234, 236-239, 344, 388-390, 393-397, 415-419,421, 421-426, 467-470, 647-651 and 758-761 from one monomer, and aminoacids 443-449 and 451 of the other monomer of human VAP-1.
 14. Thecomputer readable medium according to claim 13, wherein said bottom partof the active site cavity is lined by amino acid residues Ala370,Tyr384, Asp386, Asn470, Tpq471 and Tyr473.
 15. A synthetic, smallmolecule human VAP-1 inhibitor identifiable by a method which comprisesthe steps of a) providing atomic co-ordinates of said protein on acomputer readable medium according to claim 12, and b) using a computerto apply molecular modeling techniques to said co-ordinates.
 16. Theinhibitor according to claim 15, characterized by the formula

wherein R1 is hydrogen, lower alkyl or an optionally substituted phenylor heteroaryl group; R2 is hydrogen or lower alkyl; R3-R5 represent eachindependently hydrogen, lower alkyl, aralkyl, optionally substitutedphenyl or a heteroaryl group; R6 is naphtyl, phenyl, substituted phenylor a heteroaryl group; R7 is hydrogen, lower alkyl or aralkyl; n is 1, 2or 3; and X═O, S, SO, SO2 or NR2, with the proviso that (i) when X is O,R1 to R5 and R7 are hydrogen and n is 1, then R6 is not phenyl or2-methoxy-phenyl, (ii) when X is O, R1 is hydrogen, R2 is methyl and R3to R5 and R7 are hydrogen and n is 1, then R6 is not3-trifluoromethyl-phenyl; and (iii) when X is O, R1 is phenyl, R2 to R5and R7 are hydrogen and n is 1, then R6 is not 4-hydroxy-phthalazinyl.17. Use of a compound according to claim 15 for the manufacture of amedicament for use in treating diseases selected from the groupcomprising; acute and chronic inflammatory conditions or diseases suchas chronic arthritis, inflammatory bowel diseases, skin dermatoses andmultiple sclerosis; diseases related to carbohydrate metabolism,including diabetes and complications resulting from diabetes, e.g.,vasculopathies, such as retinopathy, nephropathy and neuropathy;aberrations in adipocyte differentiation or function, and smooth musclecell function, in particular atherosclerosis; and various vasculardiseases.
 18. A pharmaceutical composition comprising a compoundaccording to claim 15.